1. DNA Structure and Replication
Dr. Nichols Coronado HS

2. A T C G
Isn’t this a great illustration!?

3. Macromolecules: Nucleic Acids
Examples:
RNA (ribonucleic acid)
single helix
DNA (deoxyribonucleic acid)
double helix
Structure:
monomers = nucleotides
DNA
RNA

4. Nucleotides 3 parts nitrogen base (C-N ring) pentose sugar (5C)
ribose in RNA
deoxyribose in DNA
phosphate (PO4) group
Nitrogen base I’m the A,T,C,G or U part!
Are nucleic acids charged molecules?
DNA & RNA are negatively charged:
Don’t cross membranes.
Contain DNA within nucleus
Need help transporting mRNA across nuclear envelope.
Also use this property in gel electrophoresis.

5. Types of nucleotides 2 types of nucleotides different nitrogen bases
Purine = AG
Pure silver!
2 types of nucleotides
different nitrogen bases
purines
double ring N base
adenine (A)
guanine (G)
pyrimidines
single ring N base
cytosine (C)
thymine (T)
uracil (U)

6. Dangling bases? Why is this important?
Nucleic polymer
Backbone
sugar to PO4 bond
phosphodiester bond
new base added to sugar of previous base
polymer grows in one direction
N bases hang off the sugar-phosphate backbone
Dangling bases? Why is this important?

7. Pairing of nucleotides
Nucleotides bond between DNA strands
H bonds
purine :: pyrimidine
A :: T
2 H bonds
G :: C
3 H bonds
The 2 strands are complementary.
One becomes the template of the other & each can be a template to recreate the whole molecule.
Matching bases? Why is this important?

8. H bonds? Why is this important?
DNA molecule
Double helix
H bonds between bases join the 2 strands
A :: T
C :: G
H bonds = biology’s weak bond
• easy to unzip double helix for replication and then re-zip for storage
• easy to unzip to “read” gene and then re-zip for storage
H bonds? Why is this important?

9. Matching halves? Why is this a good system?
Copying DNA
Replication
2 strands of DNA helix are complementary
have one, can build other
have one, can rebuild the whole
when cells divide, they must duplicate DNA exactly for the new “daughter” cells
Why is this a good system?
Matching halves? Why is this a good system?

10. When does a cell copy DNA?
When in the life of a cell does DNA have to be copied?
cell reproduction
mitosis
gamete production
meiosis
when cells divide, they must duplicate DNA exactly for the new “daughter” cells
Why is this a good system?

11. But how is DNA copied? Replication of DNA
base pairing suggests that it will allow each side to serve as a template for a new strand

12. Models of DNA Replication
Can you design a nifty experiment to verify?
Models of DNA Replication
Alternative models
become experimental predictions
conservative
semiconservative
dispersive P 1 2

13. Semiconservative replication
1958
Semiconservative replication
Meselson & Stahl
label “parent” nucleotides in DNA strands with heavy nitrogen = 15N
label new nucleotides with lighter isotope = 14N
Make predictions…
parent
replication
15N/15N
15N parent strands

14. Semiconservative replication
1958
Semiconservative replication
Make predictions…
15N strands replicated in 14N medium
1st round of replication?
2nd round?
where should the bands be?
Matthew Stanley Meselson (b. May 24, 1930) is an American geneticist and molecular biologist whose research was important in showing how DNA replicates, recombines and is repaired in cells. In his mature years, he has been an active chemical and biological weapons activist and consultant. He is married to the medical anthropologist and biological weapons writer Jeanne Guillemin.
Dr. Franklin William Stahl (born October 8, 1929) is an American molecular biologist. With Matthew Meselson, Stahl conducted the famous Meselson-Stahl experiment showing that DNA is replicated by a semiconservative mechanism, meaning that each strand of the DNA serves as a template for the "replicated" strand.
He is Emeritus Professor of Biology[1] at the University of Oregon's Institute of Molecular Biology in Eugene, Oregon.

15. Meselson & Stahl Matthew Meselson Franklin Stahl Franklin Stahl

16. DNA Replication Origin(s) of replication Prokaryotes – Eukaryotes –
specific sequence of nucleotides recognized by replication enzymes
Prokaryotes –
Single sequence
Bidirectional Synthesis
Replication proceeds in both directions
Eukaryotes –
hundreds/thousands of origin sites per chromosome
Replication forks
Bubbles elongate as DNA is replicated and eventually fuse

17. Bidirectional Synthesis
In prokaryotes, the circular DNA is opened up, and synthesis occurs in both directions

18. Replication forks
In eukaryotes, the linear DNA has many replication forks

20. Learning Check
Break the toothpicks at the center of your models and replicate your DNA strand
You should end up with 2 complete strands of DNA
Keep in mind Chargaff’s rules and Meselson & Stahl’s semi-conservative model
Animation

21. Replication- Create a diagram that shows how the following components interact with each other (15 min)
Replication fork
RNA primer
Leading strand
DNA ligase
RNA primase
Okazaki fragments
Lagging strand
Helicase
DNA polymerase
Single stranded binding protein
Topoisomerase

22. DNA Replication Issues
1. DNA strands must be unwound during replication
DNA helicase
unwinds the strands
Single stranded binding proteins (SSB)
prevent immediate reformation of the double helix
Topoisomerases
“untying” the knots that form

23. Replication Issues
2. A new DNA strand can only elongate in the 5’  3’ direction
DNA polymerase can add only at the 3’ end
Replication is continuous on one strand
Leading Strand
discontinuous on the other
Lagging strand
Okazaki fragments

24. Okazaki fragments Synthesis of the leading strand is continuous
The lagging strand (discontinuous) is synthesized in pieces called Okazaki fragments

26. Replication Issues
3. DNA polymerase cannot initiate synthesis because it can only add nucleotides to end of an existing chain
Requires a “primer” to get the chain started
RNA Primase
can start an RNA chain from a single template strand
DNA polymerase can begin its chain after a few RNA nucleotides have been added

28. Summary
At the replication fork, the leading strand is copied continuously into the fork from a single primer
Lagging strand is copied away from the fork in short okazaki fragments, each requiring a new primer

30. Learning Check What is the purpose of DNA replication?
How is the new strand ensured to be identical to the original strand?
How is replication on one side of the strand different from the other side?

31. Replication Issues
4. Presence of RNA primer on the 5’ ends of daughter DNA leading strand leaves a gap of uncopied DNA
Repeated rounds of replication produce shorter and shorter DNA molecules
Telomeres
protect genes from being eroded through multiple rounds of DNA replication

32. Telomeres
Ends of eukaryotic chromosomes, the telomeres, have special nucleotide sequences
Humans - this sequence is typically TTAGGG, repeated ,000 times
Telomerase adds a short molecule of RNA as a template to extend the 3’ end
Room for primase & DNA pol to extend 5’ end

33. Summary
Explain how the cell overcomes each of the following issues in DNA replication
DNA strands must be unwound during replication
A new DNA strand can only elongate in the 5’  3’ direction
DNA polymerase cannot initiate synthesis and can only add nucleotides to end of an existing chain
Presence of RNA primer on the 5’ ends of daughter DNA leading strand leaves a gap of uncopied DNA